Understanding the regulatory mechanism of normal SMC development is the first step to studying congenital defects in vasculogenesis as well as the growth and control of solid tumors. In this study, the following hypothesis will be investigated: The binding of ang II to AT2-expressing cells, located in the developing tunica media, generates a paracrine factor that suppresses the growth of differentiated smooth muscle cells during vasculogenesis. Three Specific Aims have been proposed to investigate this hypothesis. In specific aim 1, the expression of AT2 by cells of the tunica media will be characterized during vascular development, SMC-specific probes will be used to determine if AT2- expressing cells are of SMC lineage. Rat aortas from embryonic day 14 to postnatal day 17 will be isolated and prepared for immunohistochemical analysis and incorporation of BrdU and Hoescht 33342. Specific Aim 2 will characterize the paracrine influence of AT2--positive cells on SMC growth by culturing proliferating SMC with condition media and cocultures of AT2--expressing cells. The identity of the putative anti-growth factor will be determined using neutralizing antibodies and inhibitors for known anti-growth factors or conditions that identify the nature of an unknown anti-growth factor. Finally, in Specific Aim 3, AT2--positive cells will be isolated using a fluorescently activated cell sorter and characterized for AT2 expression and SMC lineage using indirect immunofluorescence and quantitative RT-PCR analysis. The findings of this research will provide insight into the development of vascular SMC.